Use of phosphotyrosine phosphatase inhibitors for controlling cellular proliferation

ABSTRACT

A method of inhibiting the proliferation of B cells by using inhibitors of phosphotyrosine phosphatase can be used to regulate the immune response and to treat diseases such as leukemias or lymphomas marked by malignant proliferation of B cells. The use of such inhibitors can be combined with radiation, which produces a synergistic effect. Several types of inhibitors can be used, including: (1) compounds comprising a metal coordinate-covalently bound to an organic moiety that can form a five- or six-membered ring; (2) compounds in which vanadium (IV) is coordinate-covalently bound to an organic moiety such as a hydroxamate, α-hydroxypyridinone, α-hydroxypyrone, α-amino acid, hydroxycarbonyl, or thiohydroxamate; (3) coordinate-covalent complexes of vanadyl and cysteine or a derivative thereof; (4) nonhydrolyzable phosphotyrosine phosphatase analogues; (5) phostatin; and (6) 4-(fluoromethyl)phenyl phosphate and esterified derivatives. For the metal-containing coordinate covalent compounds, the metal is preferably vanadium (IV).

This is a Divisional of application Ser. No. 08/189,330, filed Jan. 31,1994, now U.S. Pat. No. 5,565,491, which application(s) are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

This invention is directed to the use of phosphotyrosine phosphataseinhibitors for controlling cellular proliferation, particularlyproliferation of B lymphocytes.

Tyrosine phosphorylation is known to play an essential role in thecontrol of lymphocyte function. This control is exerted by a network oftyrosine kinases and phosphotyrosine phosphatases.

Two different processes known to induce B cell apoptosis have been shownto act through tyrosine phosphorylation.

Apoptosis is a pattern of programmed cell death that involves thebreakup of the cellular DNA and can be recognized by electrophoresis ofthe DNA of the cells. When apoptosis occurs, the DNA is broken intofragments, which can be detected as a ladder on electrophoresis.

In immature B cells, stimulation of sIgM (surface immunoglobulin M) byeither antigen or anti-immunoglobulin antibodies activates the cells (G.J. V. Nossal, Annu. Rev. Immunol. 1:33-62 (1983)). Stimulation of sIg(surface immunoglobulin) on B cells induces tyrosine phosphorylation (M.R. Gold et al., Nature 345:810-813 (1990); M. A. Campbell & B. M.Sefton, EMBO J. 9:2125-2131 (1990)), which is essential for productivesIg signaling (P. J. L. Lane et al., J. Immunol. 146:715-722 (1991)). Asa result of sIg stimulation, Src family kinases are activated (A. L.Burkhardt et al., Proc. Natl. Acad. Sci. USA 88:7410-7414 (1991)).Furthermore, expression of the Src family tyrosine kinase Blk was foundto be essential in B cell lymphomas where sIgM stimulation leads togrowth arrest and apoptosis (X. R. Yao and D. W. Scott, Immunol. Rev.132:163-186 (1993)). Thus, on sIgM stimulation, tyrosine kinases such asBlk phosphorylate one or more proteins on tyrosine residues, and oncephosphorylated, these proteins are then able to induce apoptosis.However, it has also been shown that the abundant phosphotyrosinephosphatase CD45 is required for sIg signal transduction (L. B.Justement et al., Science 252:1839-1842 (1991)).

Ionizing radiation is standard therapy for B cell malignancies such asleukemias and lymphomas. It has been demonstrated that ionizingradiation stimulates B cell tyrosine kinases, triggering apoptosis andclonogenic cell death (F. M. Uckun et al., Proc. Natl. Acad. Sci. USA89:9005-9009 (1992)). In this study, the phosphotyrosine phosphataseinhibitor vanadate, administered alone, was not effective. Theactivation of tyrosine kinases by ionizing radiation was essential forthe induction of apoptosis because the tyrosine kinase inhibitorsgenistein and herbimycin A blocked the effects of the radiation.

In addition to blocking proliferation of malignant B cells in diseasessuch as leukemias and lymphomas, in a number of situations it may bedesirable to slow the growth and/or differentiation of normal B cells.Such occasions include organ transplantation, in which the immuneresponse, at least in the short term, must be suppressed. Limitedcontrol of the proliferation of B cells may also be desirable in thetreatment of autoimmune diseases such as rheumatoid arthritis and lupuserythematosus.

Accordingly, there exists a need for improved methods of controllingproliferation of B cells in malignant and non-malignant conditionswithout requiring the use of radiation. Such an approach preferablyinvolves the induction of programmed cell death (apoptosis) insusceptible cells.

SUMMARY

I have developed a method of inhibiting the proliferation of B cells byusing inhibitors of phosphotyrosine phosphatase.

Several types of inhibitors can be used:

(1) compounds comprising a metal selected from the group consisting ofvanadium (IV), copper (II) and gallium (II) coordinate-covalently boundto an organic moiety;

(2) nonhydrolyzable phosphotyrosine analogs selected from the groupconsisting of N-aryl phosphoramidates, N-aryl phosphorothioates, andN-aryl phosphonates, in which the aryl moiety is optionally substitutedat any of the ortho, meta, and para positions, and one or two of theoxygen atoms bound to the phosphorus are optionally esterified;

(3) dephostatin; and

(4) optionally esterified 4-(fluoromethyl)phenyl phosphate.

The compounds comprising vanadium (IV), copper (II), or gallium (II)coordinate-covalently bound to an organic moiety can be selected fromthe group consisting of:

(1) keto-enol tautomers with the keto and enol groups on adjacent carbonatoms that form 5-membered rings including the metal; and

(2) beta diketones in which the two keto groups are separated by onecarbon atom, that form a 6-membered ring including the metal.

For these types of metal-containing coordinate covalent compounds, themetal is preferably vanadium (IV).

When the organic moiety of the metal-containing coordinate covalentcompound is a keto-enol tautomer, the organic moiety is preferably oneof maltol, 2-hydroxy-2,4,6-cycloheptatrien-1-one,3-bromo-2-hydroxy-2,4,6-cycloheptatrien-1-one,2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one,2-hydroxy-4-methyl-2,4,6-cycloheptatrien-1-one,3-hydroxy-1,2-dimethyl-4(1H)-pyridone,3-ethyl-2-hydroxy-2-cyclopenten-1-one,3,4-dihydroxy-3-cyclobuten-1,2-dione, ethyl 2-hydroxy-4-oxo-2-pentenone,2,3,5,6-tetrahydroxy-1,4-benzoquinone,2',4'-dihydroxy-2-methoxyacetophenone,4-hydroxy-5-methyl-4-cyclopenten-1,3-dione,2-chloro-3-hydroxy-1,4-naphthoquinone,2-(4bromophenyl)-3-hydroxymaleimide,2-hydroxy-3-methyl-2-cyclopenten-1-one, 2', 3',4'-trihydroxyacetophenone, furoin, 2-hydroxy-2-methylopropiophenone,maclurin, 6-(pyrrolidinomethyl)kojic acid,alpha-acetyl-4-hydroxy-beta-(hydroxymethyl)-3-methoxycinnamic acidgamma-lactone, 4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione6-(morpholinomethyl)kojic acid,1-(4,5-dimethoxy-2-hydroxyphenyl)-3-methyl-2-buten-1-one, purpurogallin,2,3-dihydroxy-1,4-phenazinedione, alizarin orange,1-hydroxy-1-methylnaphthalen-2(1H)-one, alizarin,6-(piperidinomethyl)kojic acid, 1,2,7-trihydroxyanthraquinone,6-(4-methylpiperazinomethyl)kojic acid, fisetin,3-oxo-4,5,6-trihydroxy-3(H)-xanthene-9-propionic acid, benzoin,4'-chlorobenzoin, quercetin, morin, myricetin, or 4,4'-dimethylbenzoin.

Most preferably, the organic moiety is maltol, and the compound isbis(maltolato)oxovanadium (IV) ("BMLOV").

When the organic moiety is a beta diketone, the organic moiety ispreferably one of acetylacetone, 2-acetyl-1-tetralone, benzoylacetone,1-benzoylacetylacetone, 1,1,1-trifluoro-2,4-pentanedione,S-methyl-4,4,4-trifluoro-3-oxothiobutyrate,2-acetyl-1,3-cyclopentanedione, 3-chloro-2,4-pentanedione,1,1,1,5,5,5-hexafluoro-2,4-pentanedione,3-ureidomethylene-2,4-pentanedione, 2-acetylcyclopentanone,2-acetylcyclohexanone, 3-methyl-2,4-pentanedione, 2,4,6-heptatrione,3-ethyl-2,4-pentanedione, thenoyltrifluoroacetone,S-t-butyl-acetothioacetate, 3acetyl-5-methylhexan-2-one,3-acetyl-2-heptanone,2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione,4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione,4,4,4-trifluoro-1-phenyl-1,3-butanedione, 3-acetyl-2-octanone,1(2-hydroxy-4-methylphenyl)-1,3-butanedione,1-(2-hydroxy-5-methylphenyl)-1,3-butanedione,3-benzylidene-2,4-pentanedione,1-(2-hydroxy-5-methylphenyl)-1,3-pentanedione,2,2,6,6-tetramethyl-3,5-heptanedione,3-acetyl-5-hydroxy-2-methylchromone, (+)-3-(trifluoroacetyl)camphor,4,9-dihydro-6-methyl-5H-furo(3,2-g)(1) benzopyran-4,5,9-trione,3-(2-nitrobenzylidene)-2,4-pentanedione,1,3-bis(4-chlorophenyl)-1,3-propanedione,1,3-bis-(4-fluorophenyl)-1,3-propanedione,4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione,1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)-1,3-propanedione,2-bromo-1,3-diphenyl-1,3-propanedione, dibenzoylmethane,2-(4-chlorobenzylidene)-1-phenyl-1,3-butanedione,2(2-nitrobenzylindene)-1-phenyl-1,3-butanedione,bis(4-methoxybenzoyl)methane, or curcumin.

Most preferably, the organic moiety is acetylacetonate.

Alternatively, the coordinate covalent metal-containing compound can bea compound comprising vanadium (IV) coordinate-covalently bound to anorganic moiety of formula I ##STR1## wherein: (1) X¹ and X³ areindependently selected from the group consisting of oxygen, sulfur, andNX⁶ ;

(2) X² is selected from the group consisting of nitrogen or CX⁷ ; and

(3) X⁴, X⁵, X⁶, and X⁷ are independently selected from the groupconsisting of non-labile protons, optionally substituted alkyl groups,optionally substituted aryl groups, optionally substituted aralkylgroups, and optionally substituted alkaryl groups.

Alternatively, the organic moiety can be a compound of formula I whereinat least one pair of X⁴ to X⁷ together with the intervening atoms,represents an optionally substituted, saturated or unsaturatedhomocyclic or heterocyclic ring.

In another alternative, the organic moiety can be a compound of formulaI wherein X¹ is a NX⁶ group, X⁴ is a group X⁸ H where X⁸ is selectedfrom the group consisting of oxygen or sulfur, and wherein one protonattached to X¹ or X⁸ is labile.

Suitable compounds of formula I include:

(1) a hydroxamate of formula II; ##STR2## (2) a α-hydroxypyridione offormula III; ##STR3## (3) a α-hydroxyprone of formula IV; ##STR4## (4)an α-amino acid of formula V; ##STR5## (5) a hydroxycarbonyl of formulaVI or formula VII; and ##STR6## (6) a thiohydroxamate of formula VIII orformula IX, wherein R¹ to R¹⁹ are hydrogen or optionally substitutedalkyl. ##STR7##

In these formulas, R¹ to R¹⁹ are hydrogen or optionally substituted C₁-C₄ alkyl, e.g., hydroxylated alkyl.

Alternatively, the organic compound can be a coordinate-covalent complexof vanadyl and cysteine or a derivative thereof of formula X ##STR8##wherein n, p, and m are integers equal to 1 or 2 respectively, and: (1)when p is equal to 1, then Y is selected from the group consisting of ahydrogen atom and a R'--CO group; and

(a) when n is equal to 1 and m is equal to 2, X is selected from thegroup consisting of an OH group, an OR group, and an NHR group wherein Ris selected from the group consisting of an alkyl group comprising from2 to 9 carbon atoms, an aryl group, or an aralkyl group, wherein, when Xis an OH group, Y is a R'--CO group wherein R' is selected from thegroup consisting of an alkyl group comprising from 2 to 9 carbon atoms,and, when X is selected from the group consisting of an OR group and aNHR group, Y is H;

(b) when n is equal to 2 and m is equal to 1, X is selected from thegroup consisting of a difunctional amine of formula WC CH₂ NH--!₂, adifunctional alcohol of formula WC CH₂ O--!₂, and a difunctionalamine-alcohol of formula WC(CH₂ NH--)(CH₂ O--), wherein W is an alkylgroup of from 2 to 9 carbon atoms other than butyl; and

(2) when p is equal to 2, then n is equal to 1, m is equal to 1, X is anOH group, and Y is selected from the group consisting of ZCH(CO--)₂,--CH₂ --, or ZCH(CH₂ --)₂ in which Z is an alkyl, aryl, or aralkylgroup.

Suitable organic moieties of this type include:

(1) a compound of formula XI; ##STR9## (2) a compound of formula XII;##STR10## (3) a compound of formula XIII; ##STR11## (4) a compound offormula XIV; and ##STR12## (5) a compound of formula XV. ##STR13##

When the phosphotyrosine phosphatase inhibitor is nonhydrolyzablephosphotyrosine analog, it can be a N-aryl phosphoramidate of formulaXVI, in which each of R₁ through R₇ is selected from the groupconsisting of hydrogen and C₁ -C₅ alkyl. ##STR14##

Alternatively, it can be a phosphorothioate of formula XVII in whicheach of R₁ through R₇ is selected from the group consisting of hydrogenand C₁ -C₅ alkyl. ##STR15##

In another alternative, it can be a phosphonate of formula XVIII inwhich each of R₁ through R₇ is selected from the group consisting ofhydrogen and C₁ -C₅ alkyl. ##STR16##

For phosphoramidates of formula XVI, phosphorothioates of formula XVII,or phosphonates of formula XVIII, typically each of R₁ through R₅ ishydrogen. In that case, at least one of R₆ and R₇ is preferably otherthan hydrogen.

Another phosphotyrosine phosphatase inhibitor useful in the methods ofthe present invention is dephostatin.

Still other phosphotyrosine phosphatase inhibitors are useful in themethods of the present invention, including optionally esterified4-(fluoromethyl)phenyl phosphates of formula XX, in which R₁ and R₂ areindependently selected from the group consisting of hydrogen and C₁ -C₅alkyl. ##STR17##

A method for inhibiting B cell proliferation according to the inventioncomprises the step of contacting proliferating B cells with aphosphotyrosine phosphatase inhibitor as described above, thephosphotyrosine phosphatase inhibitor being administered in a quantitysufficient to detectably inhibit proliferation as measured byincorporation of nucleotides into DNA.

A method of inhibiting phosphotyrosine phosphatase in proliferating Bcells according to the invention comprises the step of contactingproliferating B cells with a phosphotyrosine phosphatase inhibitor asdescribed above, the inhibitor being administered to the B cells in aquantity sufficient to inhibit the activity of phosphotyrosinephosphatase in the cells.

Compositions and methods according to the present invention are alsouseful for studying and/or modifying signaling in T cells, particularlysignals involving CD28.

The invention further includes a method of treating a malignantproliferative disorder selected from the group consisting of leukemiasand lymphomas wherein the proliferating cells are selected from thegroup consisting of B cells and myeloid cells. The method comprises thestep of contacting the proliferating malignant cells with aphosphotyrosine phosphatase inhibitor as described above, the compoundbeing administered in a quantity sufficient to significantly inhibitproliferation of the malignantly proliferating cells. The method canfurther include a step of delivering ionizing radiation to the cellscontacted with the phosphotyrosine phosphatase inhibitor. The ionizingradiation is delivered in a dose sufficient to induce a substantialdegree of cell killing among the malignantly proliferating cells. Thedegree of cell killing induced is substantially greater than thatinduced by either the coordinate covalent metal-organic compound or theionizing radiation alone.

Another aspect of the present invention is a method of preventing theclass-switching of antibody-producing cells. The method comprisesadministering to antibody-producing cells a quantity of aphosphotyrosine phosphatase inhibitor sufficient to detectably reducethe production of IgE antibody by the cells. Typically, the cells alsoproduce IgG antibody, and the quantity of the phosphotyrosinephosphatase inhibitor is such that the ratio of the quantity of IgGantibody produced by the cells to the quantity of IgE antibody producedby the cells increases. A preferred phosphotyrosine phosphataseinhibitor for preventing class-switching is BMLOV.

Yet another aspect of the present invention is novel phosphotyrosinephosphatase inhibitors, including vanadyl 2-acetyl-1-tetralone, vanadyl2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one.

Additional novel phosphotyrosine phosphatase inhibitors according to thepresent invention include metal-organic covalent compounds comprisingvanadium (IV) coordinate-covalently bound to an organic moiety that is aketo-enol tautomer with the keto and enol groups on adjacent carbonatoms and that forms a 5-membered ring including the metal, the organicmoiety being selected from the group consisting of2-hydroxy-2,4,6-cycloheptatrien-1-one,3-bromo-2-hydroxy-2,4,6-cycloheptatrien-1-one,3-hydroxy-1,2-dimethyl-4(1H)-pyridone,3-ethyl-2-hydroxy-2-cyclopenten-1-one,3,4-dihydroxy-3-cyclobuten-1,2-dione, ethyl 2-hydroxy-4-oxo-2-pentenone,2,3,5,6-tetrahydroxy-1,4-benzoquinone,2',4'-dihydroxy-2-methoxyacetophenone,4-hydroxy-5-methyl-4-cyclopenten-1,3-dione,2-chloro-3-hydroxy-1,4-naphthoquinone,2-(4-bromophenyl)-3-hydroxymaleimide,2-hydroxy-3-methyl-2-cyclopenten-1-one, 2',3',4'-trihydroxyacetophenone,furoin, 2-hydroxy-2-methylpropiophenone, maclurin,alpha-acetyl-4-hydroxy-beta-(hydroxymethyl)-3-methoxycinnamic acidgamma-lactone, 4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione,1-(4,5-dimethoxy-2-hydroxyphenyl)-3-methyl-2-buten-1-one, purpurogallin,2,3-dihydroxy-1,4-phenazinedione, alizarin orange,1-hydroxy-1-methylnaphthalen-2(1H)-one, alizarin,1,2,7-trihydroxyanthraquinone, fisetin,3-oxo-4,5,6-trihydroxy-3(H)-xanthene-9-propionic acid, benzoin,4'-chlorobenzoin, quercetin, morin, myricetin, and 4,4'-dimethylbenzoin.

Additional novel phosphotyrosine phosphatase inhibitors according to thepresent invention further include metal-organic covalent compoundscomprising vanadium (IV) coordinate-covalently bound to an organicmoiety that is a beta diketone in which the two keto groups areseparated by one carbon atom that forms a 6-membered ring including themetal, the organic moiety being selected from the group consisting of2-acetyl-1-tetralone, 1-benzoylacetylacetone,1,1,1-trifluoro-2,4-pentanedione,S-methyl-4,4,4-trifluoro-3-oxothiobutyrate,2-acetyl-1,3-cyclopentanedione, 3-chloro-2,4-pentanedione,1,1,1,5,5,5-hexafluoro-2,4-pentanedione,3-ureidomethylene-2,4-pentanedione, 2-acetylcyclopentanone,2-acetylcyclohexanone, 3-methyl-2,4-pentanedione, 2,4,6-heptatrione,3-ethyl-2,4-pentanedione, thenoyltrifluoroacetone,S-t-butyl-acetothioacetate, 3-acetyl-5-methylhexan-2-one,3-acetyl-2-heptanone,2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione,4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione,4,4,4-trifluoro-1-phenyl-1,3-butanedione, 3-acetyl-2-octanone,1(2-hydroxy-4-methylphenyl)-1,3-butanedione,1-(2-hydroxy-5-methylphenyl)-1,3-butanedione,3-benzylidene-2,4-pentanedione,1-(2-hydroxy-5-methylphenyl)-1,3-pentanedione,2,2,6,6-tetramethyl-3,5-heptanedione,3-acetyl-5-hydroxy-2-methylchromone, (+)-3-(trifluoroacetyl)camphor,4,9-dihydro-6-methyl-5H-furo(3,2-g)(1) benzopyran-4,5,9-trione,3-(2-nitrobenzylidene)-2,4-pentanedione,1,3-bis(4-chlorophenyl)-1,3-propanedione,1,3-bis-(4-fluorophenyl)-1,3-propanedione,4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione,1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)-1,3-propanedione,2-bromo-1,3-diphenyl-1,3-propanedione, dibenzoylmethane,2-(4-chlorobenzylidene)-1-phenyl-1,3-butanedione,2-(2-nitrobenzylidene)-1-phenyl-1,3-butanedione, bis(4-methoxybenzoyl)methane, and curcumin.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1A is a photograph of an anti-phosphotyrosine western blotfollowing sodium dodecyl sulfate-polyacrylamide electrophoresis of celllysates from Ramos cells, a human B cell lymphoma cell line, aftertreatment with bis(maltolato)oxovanadium (IV) (BMLOV), showing the dosedependence of the resulting phosphorylation after a one-hour exposure toBMLOV;

FIG. 1B is a similar photograph of an anti-phosphotyrosine western blotshowing the effects of treating the cells with 100 μM BMLOV for varyingtimes;

FIG. 1C is a similar photograph of an anti-phosphotyrosine western blot,showing the high levels of phosphorylation reached after exposure to 50μM BMLOV for 16 hours;

FIG. 2A is a photograph of a stained electropherogram on an agarose gelof DNA from Ramos cells and human promyelocytic leukemia HL-60 cellsafter treatment of the cells with BMLOV, showing the breakdown of theDNA into fragments characteristic of apoptosis;

FIG. 2B is a similar photograph of a stained electropherogram of DNA onan agarose gel of DNA from the human T cell leukemia cell lines Jurkatand CEM, and the human colon carcinoma cell line 3347, after treatmentof the cells with BMLOV, showing that apoptosis did not occur,demonstrating the selectivity of BMLOV;

FIG. 3 is a graph showing the effects of varying doses of BMLOV onthymidine incorporation in normal tonsillar cells stimulated withvarying combinations of ligands;

FIG. 4 is a similar graph showing the effects of varying doses of BMLOVon thymidine incorporation with tonsillar cells from a second donor;

FIG. 5 is a graph showing the results of clonogenic assays aftertreatment of Ramos B cells with radiation alone, BMLOV alone, orradiation and BMLOV;

FIG. 6 is a graph showing the inhibition by BMLOV of growth ofperipheral blood lymphocytes (PBL) driven by anti-CD28 antibody plusinterleukin-2;

FIG. 7 is a graph showing the inhibition by BMLOV of growth ofperipheral blood lymphocytes (PBL) driven by anti-CD28 antibody plusanti-CD3 antibody;

FIG. 8 is a graph showing the inhibition by BMLOV of growth in mixedlymphocyte response cultures dependent on CD28 costimulation;

FIG. 9 is a graph showing the inhibition of pp60^(c-Src) kinase activityin two colon carcinoma cell lines expressing Src by BMLOV;

FIG. 10 is a graph showing the results of fluorescence-activated cellsorting (FACS) analysis of the cell cycle stage of Ramos B cells aftertreatment with BMLOV; and

FIG. 11 is a photograph of an anti-phosphotyrosine western blot similarto those in FIGS. 1A-1C showing the effects of treating the cells withvanadyl acetylacetonate and analogs of vanadyl acetylacetonate in whichthe vanadium is replaced by molybdenum, chromium, iron, manganese, orcopper, showing the induction of phosphorylation by vanadylacetylacetonate in a pattern similar to that of BMLOV.

DESCRIPTION

I have developed an effective means of inhibiting phosphotyrosinephosphatase, particularly in B cells, as well as novel vanadyl compoundspossessing inhibitory activity for phosphotyrosine phosphatase.

The inhibition of phosphotyrosine phosphatase can be used to inhibit theproliferation of both malignant and normal B cells, as well as otherphysiological functions depending on the balance between phosphorylatedand dephosphorylated tyrosine residues.

I. PHOSPHOTYROSINE PHOSPHATASE INHIBITORS

In general, two types of phosphotyrosine phosphatase inhibitors areuseful in the methods of the present invention. These are metal-organiccoordinate covalent compounds and nonhydrolyzable phosphotyrosineanalogs. Other phosphotyrosine phosphatase inhibitors are also useful inthe method of the present invention.

A. Metal-Organic Coordinate Covalent Compounds

Metal-organic coordinate covalent compounds useful in the methods of thepresent invention comprise a metal selected from the group consisting ofvanadium (IV), copper (II) and gallium (II) coordinate-covalently boundto an organic moiety that can be either: (1) a keto-enol tautomer withthe keto and enol groups on adjacent carbon atoms and that forms a5-membered ring including the metal; and (2) a beta diketone in whichthe two keto groups are separated by one carbon atom that forms a6-membered ring including the metal.

The metal is preferably vanadium (IV). Other metals can give differentpatterns of inhibition in particular cell types.

If the organic moiety is a keto-enol tautomer forming a 5-membered ringincluding the metal, it is preferably one of the following moieties:maltol, kojic acid, 2-hydroxy-2,4,6-cycloheptatrien-1-one,3-bromo-2-hydroxy-2,4,6-cycloheptatrien-1-one,2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one,2-hydroxy-4-methyl-2,4,6-cycloheptatrien-1-one,3-hydroxy-1,2-dimethyl-4(1H)-pyridone,3-ethyl-2-hydroxy-2-cyclopenten-1-one,3,4-dihydroxy-3-cyclobuten-1,2-dione, ethyl 2-hydroxy-4-oxo-2-pentenone,2,3,5,6-tetrahydroxy-1,4-benzoquinone,2',4'-dihydroxy-2-methoxyacetophenone,4-hydroxy-5-methyl-4-cyclopenten-1,3-dione,2-chloro-3-hydroxy-1,4-naphthoquinone,2-(4-bromophenyl)-3-hydroxymaleimide,2-hydroxy-3-methyl-2-cyclopenten-1-one, 2',3',4'-trihydroxyacetophenone,furoin, 2-hydroxy-2-methylpropiophenone, maclurin,6-(pyrrolidinomethyl)kojic acid,alpha-acetyl-4-hydroxy-beta-(hydroxymethyl)-3-methoxycinnamic acidgamma-lactone, 4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione,6-(morpholinomethyl)kojic acid,1-(4,5-dimethoxy-2-hydroxyphenyl)-3-methyl-2-buten-1-one, purpurogallin,2,3-dihydroxy-1,4-phenazinedione, alizarin orange,1-hydroxy-1-methylnaphthalen-2(1H)-one, alizarin,6-(piperidinomethyl)kojic acid, 1,2,7-trihydroxyanthraquinone,6-(4-methylpiperazinomethyl)kojic acid, fisetin,3-oxo-4,5,6-trihydroxy-3(H)-xanthene-9-propionic acid, benzoin,4'-chlorobenzoin, quercetin, morin, myricetin, or 4,4'-dimethylbenzoin.More preferably, the organic moiety is maltol, and the resultingcompound is bis(maltolato)oxovanadium (IV) ("BMLOV").

If the organic moiety is a beta diketone, the organic moiety ispreferably one of the following moieties: acetylacetone,2-acetyl-1-tetralone, benzoylacetone, 1-benzoylacetylacetone,1,1,1-trifluoro-2,4-pentanedione,S-methyl-4,4,4-trifluoro-3-oxothiobutyrate,2-acetyl-1,3-cyclopentanedione 3-chloro-2,4-pentanedione,1,1,1,5,5,5-hexafluoro-2,4-pentanedione,3-ureidomethylene-2,4-pentanedione, 2-acetylcyclopentanone,2-acetylcyclohexanone, 3-methyl-2,4-pentanedione, 2,4,6-heptatrione,3-ethyl-2,4-pentanedione, thenoyltrifluoroacetone,S-t-butyl-acetothioacetate, 3-acetyl-5-methylhexan-2-one,3-acetyl-2-heptanone,2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione,4-hydroxy-5-phenyl-4-cyclopenten-1,3-dione,4,4,4-trifluoro-1-phenyl-1,3-butanedione, 3-acetyl-2-octanone,1(2-hydroxy-4-methylphenyl)-1,3-butanedione,1-(2-hydroxy-5-methylphenyl)-1,3-butanedione,3-benzylidene-2,4-pentanedione,1(2-hydroxy-4-methylphenyl)-1,3-pentanedione,2,2,6,6-tetramethyl-3,5-heptanedione,3-acetyl-5-hydroxy-2-methylchromone, (+)-3-(trifluoroacetyl)camphor,4,9-dihydro-6-methyl-5H-furo(3,2-g)(1)benzopyran-4,5,9-trione,3-(2-nitrobenzylidene)-2,4-pentanedione,1,3-bis(4-chlorophenyl)-1,3-propanedione,1,3-bis-(4-fluorophenyl)-1,3-propanedione,4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione,1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)-1,3-propanedione,2-bromo-1,3-diphenyl-1,3-propanedione, dibenzoylmethane,2-(4-chlorobenzylidene)-1-phenyl-1,3-butanedione,2-(2-nitrobenzylidene)-1-phenyl-1,3-butanedione,bis(4-methoxybenzoyl)methane, and curcumin. Preferably, the organicmoiety is acetylacetone, and the resulting compound is vanadylacetylacetonate.

Among novel compounds containing vanadium coordinate-covalently bound toan organic moiety that are believed to have activity as phosphotyrosinephosphatase inhibitors are the compounds vanadyl 2-acetyl-1-tetralone,vanadyl 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one, and vanadyl2-hydroxy-4-methyl-2,4,6-cycloheptatrien-1-one.

Other metal-organic coordinate covalent compounds useful for theprocesses of the present invention include complexes in which the metalis preferably vanadium and the organic moiety is one of formulas Ithrough IX, of which formula I is a general formula and formulas IIthrough IX represent particular classes of compounds of formula I.##STR18##

In formula I, X¹ and X³ are independently oxygen, sulfur, or NX⁶,preferably oxygen or NX⁶. X² is nitrogen or CX⁷. X⁴, X⁵, X⁶, and X⁷ areindependently non-labile protons or optionally substituted alkyl, aryl,aralkyl or alkaryl. Alternatively, at least one pair of X⁴ to X⁷,preferably X⁴ and X⁵, together with the intervening atoms can representan optionally substituted, saturated or unsaturated homocyclic orheterocyclic ring. Alternatively, where X¹ is a NX⁶ group, X⁴ canrepresent a group X⁸ H where X⁸ is oxygen or sulfur, and one protonattached to X¹ or X⁸, preferably a proton attached to X¹, is labile.

Typical organic moieties of formula I are α-amino acids (other thancysteine), hydroxamates, thiohydroxamates, α-hydrozycarbonyls such asα-hydroxypyridinones or α-hydroxypyrones.

When the organic moiety comprises a homocyclic or heterocyclic ring, thering is preferably a 5-, 6-, or 7-membered ring. If the ring isheterocyclic, it can contain 1, 2, or 3 heteroatoms, typically 1. Theheteroatoms are selected from O, N, and S, and are preferably O or N.Each aryl group is preferably phenyl or naphthyl, typically phenyl. Eachalkyl group or moiety contains 1 to 6 carbon atoms, typically 1 to 4.The optional substituents, which do not include thiol groups, arepreferably selected from hydroxy, alkoxy, oxo, amide, and amine groups,as well as alkyl groups carrying such substituents. These groups can beselected for their ability to enhance the hydrophilicity orlipophilicity of the complex or to enable the complex to be conjugatedto another molecule such as a protein, a polymer, or anotherbiologically active molecule.

Particularly suitable organic moieties include the hydroxamates offormula II, the α-hydroxypyridinones of formula III, theα-hydroxypyrones of formula IV, the α-amino acids of formula V, thehydroxycarbonyls of formulas VI and VII, and the thiohydroxamates offormulas VIII and IX. In these formulas, R¹ to R¹⁹ are hydrogen oroptionally substituted, e.g., hydroxylated, C₁ to C₄ alkyl.

Still other metal-organic coordinate covalent compounds are useful inprocesses according to the present invention. These coordinate covalentcompounds are complexes of vanadyl and cysteine or cysteine derivativesand have the general structure shown in formula X: ##STR19##

In formula X, either x is 1 and y is 0 or x is 0 and y is 1. The valuesof n, p, and m are 1 or 2. The cysteine moiety can be D-cysteine orL-cysteine.

When p is equal to 1, then Y is selected from the group consisting of ahydrogen atom and a R'--CO group.

When n is equal to 1 and m is equal to 2, X is selected from the groupconsisting of an OH group, an OR group, and an NHR group wherein R isselected from the group consisting of an alkyl group comprising from 2to 9 carbon atoms, an aryl group, or an aralkyl group. In thisstructure, when X is an OH group, Y is a R'--CO group wherein R' isselected from the group consisting of an alkyl group comprising from 2to 9 carbon atoms, and, when X is selected from the group consisting ofan OR group and a NHR group, Y is H.

When n is equal to 2 and m is equal to 1, X is selected from the groupconsisting of a difunctional amine of formula WC CH₂ NH--!₂, adifunctional alcohol of formula WC CH₂ O--!₂, and a difunctionalamine-alcohol of formula WC(CH₂ NH--) (CH₂ O--), wherein W is an alkylgroup of from 2 to 9 carbon atoms.

when p is equal to 2, then n is equal to 1, m is equal to 1, X is an OHgroup, and Y is selected from the group consisting of ZCH(CO--)₂, --CH₂--, or ZCH(CH₂ --)₂ in which Z is an alkyl, aryl, or aralkyl group.

One class of compounds of this type has x equal to 1, y equal to 0, andp equal to 1. In this class of compounds, Y is hydrogen and Z is a minuscharge. This class of compounds has a free amine group that is bound tothe vanadyl; the ligand bound to the vanadyl is bimolecular, in that twoorganic moieties are complexed to a single vanadyl ion. The ligand has anegative charge on a sulfur atom. This class of compounds is depicted informula XI. ##STR20##

In formula XI, X is an --OR group or a --NHR (amine) group, in which theR moiety is an aryl or aralkyl group or an alkyl group other thanmethyl. Where X is a --NHR group, the compound is a complex of vanadylwith an amide of cysteine such as a butylamide of cysteine or anoctylamide of cysteine. The vanadyl-octylamide complex of L-cysteine isshown in formula XIa. ##STR21##

Where X is an --OR group, the compound is, for example, a complex ofvanadyl with an ester of the cysteine, such as an octylester of cysteineor a butylester of cysteine.

Another class of compounds of this type has n equal to 2 and m equalto 1. In this class of compounds, the ligand is monomolecular. Thisclass of compounds is depicted in formula XII. ##STR22##

In formula XII, X corresponds to a difunctional amine of formula WC CH₂NH--!₂, a difunctional alcohol of formula WC CH₂ O--!₂, or adifunctional amine-alcohol of formula WC(CH₂ NH--)(CH₂ O--). R is analkyl group of from 2 to 9 carbon atoms.

Yet another class of compounds of this type has x equal to 0,1 y equalto 1, and n equal to 1. In this class of compounds, X is a O⁻ group andZ is hydrogen. This class of compounds includes a bimolecular ligandcomprising a free carboxyl group and is depicted in formula XIII.##STR23##

In molecules of formula XIII, Y corresponds to a R--CO (ketone) group inwhich the R moiety is an alkyl, aryl, or aralkyl group.

Yet another class of compounds of this type has p equal to 2 and m equalto 1. This class of compounds includes a monomolecular ligand comprisingtwo free carboxyl groups and is depicted in formula XIV. ##STR24##

In molecules of formula XIV, Y corresponds to a group of formulaZCH(CO--)₂, --CH₂ --, or ZCH(CH₂ --)₂ in which Z is an alkyl, aryl, oraralkyl group.

Still another class of compounds of this type has x equal to 0, y equalto 1, Y a CH₂ group, and Z a minus charge. In this class of compounds, nand p are each 1 and m is 2. This class of compounds includes amonomolecular ligand with substituted amino and carboxyl groups and isdepicted in formula XV. ##STR25##

In formula XV, X is an --OR group or a --NHR (amine) group, in which theR moiety is an aryl or aralkyl group or an alkyl group other thanmethyl.

B. Nonhydrolyzable Phosphotyrosine Analogs

Another class of phosphotyrosine phosphatase inhibitors useful in themethods of the present invention is nonhydrolyzable phosphotyrosineanalogs. These analogs can be either: (1) N-aryl phosphoramidates; (2)N-aryl phosphorothioates; or (3) N-aryl phosphonates. The N-arylphosphoramidates have the structure shown in formula XVI, the N-arylphosphorothioates have the structure shown in formula XVII, and theN-aryl phosphonates have the structure shown in formula XVIII. ##STR26##

In either the N-aryl phosphoramidates, the N-aryl phosphorothioates, orthe N-aryl phosphonates, the aryl moiety can be optionally substitutedat any of the ortho, meta, and/or para positions. Similarly, one or twoof the oxygen atoms bound to the phosphorus are optionally esterified.

In the N-aryl phosphoramidate of formula XVI, each of R₁ through R₇ canbe selected from the group consisting of hydrogen and C₁ -C₅ alkyl,which can be either straight-chain or branched-chain. Preferably, eachof R₁ through R₅ is hydrogen, hydrogen. Preferably, when each of R₁through R₅ is hydrogen, at least one of R₆ and R₇ is other thanhydrogen.

When the nonhydrolyzable phosphotyrosine analog is a phosphorothioate offormula XVII, preferably each of R₁ through R₇ is hydrogen or C₁ -C₅alkyl, which can be either straight-chain or branched-chain. Preferably,each of R₁ through R₅ is hydrogen. When each of R₁ through R₅ ishydrogen, at least one of R₆ and R₇ is other than hydrogen.

Similarly, when the nonhydrolyzable phosphotyrosine analogue is aphosphonate of formula XVIII, preferably each of R₁ through R₇ ishydrogen or C₁ -C₅ alkyl, which can be either straight-chain orbranched-chain. Preferably, each of R₁ through R₅ is hydrogen. When eachof R₁ through R₅ is hydrogen, at least one of R₆ and R₇ is other thanhydrogen.

C. Additional Phosphotyrosine Phosphatase Inhibitors

Additional phosphotyrosine phosphatase inhibitors exist that are usefulin methods according to the present invention. These additionalphosphotyrosine phosphatase inhibitors include dephostatin and4-(fluoromethyl)phenyl phosphate and its esterified derivatives.

1. Dephostatin

Dephostatin is a phosphotyrosine phosphatase inhibitor isolated fromStreptomyces sp. MJ742-NF5 (M. Imoto et al., "Dephostatin, a NovelProtein Tyrosine Phosphatase Inhibitor Produced by Streptomyces. I.Taxonomy, Isolation, and Characterization," J. Antibiotics 46:1342-1346(1993)). It has structure XIX shown below ##STR27## and is a competitiveinhibitor of phosphotyrosine phosphatase, competing with the substratefor the enzyme. Dephostatin can be extracted from the broth filtrate ofa Streptomyces culture with ethyl acetate and purified by silica gelchromatography and high-pressure liquid chromatography (HPLC).

2. 4-(Fluoromethyl)Phenyl Phosphate and Its Esterified Derivatives

The inhibitor of human prostatic acid phosphatase 4-(fluoromethyl)phenylphosphate (formula XX; R₁ and R₂ each H) (J. K. Myers & T. S. Widlanski,"Mechanism-Based Inactivation of Prostatic Acid Phosphatase," Science262:1451-1453 (1993)), together with its esterified derivatives, arealso phosphotyrosine phosphatase inhibitors that are useful in processesaccording to the present invention. In compounds of formula XX useful inprocesses according to the present invention, R₁ and R₂ are eitherhydrogen or C₁ -C₅ alkyl, which can be either straight-chain orbranched-chain. Preferably, at least one of R₁ and R₂ is C₁ -C₅ alkyl.##STR28## D. Synthesis of Compounds

1. Synthesis of Metal-Organic Coordinate Covalent Compounds

In general, the metal-organic coordinate covalent compounds aresynthesized by the general method described forbis(maltolato)oxovanadium (IV) in J. H. McNeill et al.,"Bis(maltolato)oxovanadium (IV) Is a Potent Insulin Mimic," J. Med.Chem. 35:1489-1491 (1992). In general, the compounds are prepared bycombining the organic ligand (maltol for BMLOV) and vanadyl sulfate in a2:1 ratio, raising the pH of the solution to 8.5, refluxing overnight,and collecting the compound that precipitates upon cooling.

In the generalization of this method, the organic moiety is dissolved inwater at alkaline pH. Depending on the compound, a higher pH may berequired for solubilization. For some compounds, a water-miscible, lesspolar solvent might need to be added to dissolve the compound. Suchsolvents can include aprotic solvents such as acetonitrile,dimethylsulfoxide, or dimethylformamide, although other suitablesolvents are also known in the art. Vanadyl sulfate, or another metalsalt if desired, is then added in a sufficient quantity to achieve a 2:1molar ratio between the organic moiety and the metal salt. The solutionis then heated with a condenser attached to the reaction flask to permitrefluxing. Following the reaction, the solution is then cooled and theproduct is recovered as a precipitated solid. In some cases, the productmay not precipitate, and can then be recovered by rotary evaporation ofthe solvent or by other means, such as chromatography.

Vanadium chelates with ligands of Formula I can be prepared in a one-potsynthesis analogous to that described for gallium, aluminum, or indiumcomplexes by Zhang et al., Can. J. Chem., 67:1708-1710 (1989).

In general, the compounds of formula X are synthesized by the followingprocess:

(1) reacting a mono- or bifunctional amine or mono- or bifunctionalalcohol, or an amine alcohol, with cysteine or a derivative thereof,which is protected on the amine function and on the thiol function by at-butyloxycarbonyl group in the presence ofdicyclohexylcarbodiimide/hydroxybenzotriazole;

(2) eliminating the butyloxycarbonyl group by acidolysis;

(3) adding a vanadyl sulfate dissolved in water under a nitrogenatmosphere to the hydrochloride of the cysteine derivative in adimethylformamide-borate buffer mixture at a pH of about 10, with acysteine:vanadyl ratio of about 5:1;

(4) recovering the precipitated complex; and

(5) washing the recovered precipitate with water and drying theprecipitate.

A second method for preparing compounds according to formula I havingone free amine group or substituted amine and carboxyl groups comprises:

(1) reacting the cysteine or a derivative thereof with vanadyl sulfateat a pH of about 7 in water;

(2) recovering the complex obtained after evaporation;

(3) redissolving the complex in dimethylformamide;

(4) coupling with a mono- or bifunctional amine, a mono- or bifunctionalalcohol, or an amine-alcohol, in the presence ofdimethylaminopropylethylcarbodiimide; and

(5) recovering the complex after vacuum evaporation of the solvent, andwashing the complex with ether and with water.

In general, a method for preparing the compounds of formula XI or XVcomprises:

(1) reacting a monofunctional amine or a monofunctional alcohol with thecysteine or a derivative thereof protected on the amine function and onthe thiol function by a t-butyloxycarbonyl group in the presence ofdicyclohexylcarbodiimide/hydroxybenzotriazole;

(2) eliminating the t-butyloxycarbonyl group by acidolysis with the aidof hydrochloric acid and dioxane;

(3) adding vanadyl sulfate which is dissolved in water, under a nitrogenatmosphere, to the solution of the hydrochloride of the cysteinederivative in a dimethylformamide-borate buffer mixture at a pH of about10 with a cysteine-vanadyl ratio of 5:1;

(4) stirring the mixture for two hours in a nitrogen atmosphere;

(5) recovering the complex formed by precipitation by filtration; and

(6) washing with water and drying the precipitate.

Another method for preparing the compounds of formula XI or XVcomprises:

(1) reacting the cysteine or derivative thereof with vanadyl sulfate ata pH of about 7 in water;

(2 ) recovering the complex obtained after evaporation;

(3 ) redissolving the complex formed in dimethyl formamide as above;

(4) coupling with a monofunctional amine or a monofunctional alcohol inthe presence of dimethylaminopropylethylcarbodiimide; and

(5) recovering the complex after vacuum evaporation of the solvent,washing with ether and with water, dissolving the product in methanol,evaporation, and reprecipitation by ethyl ether.

The preparation of the compound of formula XII comprises reacting abifunctional amine or a bifunctional alcohol or an amine-alcohol withthe cysteine or a derivative thereof. The remaining procedure isidentical to that described above for the preparation of compounds XIand XV.

The preparation of the compound of formula XIII where Y corresponds tothe RCO-- group comprises:

(1) coupling an activated derivative (an ester or an acyl chloride) ofthe RCOOH acid with a cysteine previously protected on its thiolfunction by a t-butyloxycarbonyl group;

(2) deprotecting the thiol function by acidolysis; and

(3) complexing the resulting N-acylated derivative with the vanadylsulfate in a DMF-water medium at a pH of 10.

A method for preparing the compound of formula XIV where Y correspondsto RCH(CO--)₂, comprises:

(1) coupling an activated derivative (ester or acyl chloride) of thediacid RCH(COOH)₂ with a cysteine previously protected on its thiolfunction with a t-butyloxycarbonyl group;

(2) deprotecting the thiol function by acidolysis;

(3) reducing the. N-acylated derivative thus obtained; and

(4) complexing the reduced derivative in a DMF-water medium at a pH of10 in the presence of vanadyl sulfate.

2. Synthesis of Nonhydrolyzable Phosphotyrosine Analogs

The nonhydrolyzable phosphotyrosine analogs, which are phosphoramidates,phosphorothioates, or phosphonates, can be synthesized by methods wellunderstood in the art for synthesis of these compounds.Phosphorothioyltyrosine can be synthesized by sulfurization of theintermediate phosphite triester using phenylacetyl disulfide (D.B.A.Debont et al., "Solid-Phase Synthesis ofO-Phosphothioylserine-Containing and O-Phosphorothreonine-ContainingPeptides as Well as of O-Phosphoserine-Containing andO-Phosphothreonine-Containing Peptides," J. Org. Chem. 58:1309-1317(1993)). Phosphonates of tyrosine can be synthesized with the use of thereagent 4-(di-t-butylphosphono)methyl!-N-(fluoren-9-ylmethoxycarbonyl)-D,L-phenylalanine(T. R. Burke et al., "Preparation ofFluoro-4-(Phosphonomethyl)-D,L-Phenylalanine andHydroxy-4-(Phosphonomethyl)-D,L-Phenylalanine Suitably Protected forSolid-Phase Synthesis of Peptides Containing Hydrolytically StableAnalogues of O-Phosphotyrosine," J. Org. Chem. 58:1336-1340 (1993)).

II. USE OF PHOSPHOTYROSINE PHOSPHATASE INHIBITORS

Phosphotyrosine phosphatase inhibitors, including metal-organiccoordinate covalent compounds such as vanadyl compounds andnonhydrolyzable phosphotyrosine analogs, can be used to block or alter anumber of processes involving phosphotyrosine metabolism.

A. Inhibition of Proliferation of B Cells

In B cells, the level of tyrosine phosphorylation is used for metabolicregulation. Accumulation of an excessive level of tyrosinephosphorylation, such as by the continued activity of tyrosine kinasesin the absence of significant phosphotyrosine phosphatase activity,leads to apoptosis, which is programmed cell death marked byfragmentation of cellular DNA. Therefore, the administration ofinhibitors of phosphotyrosine phosphatase can be used to control theproliferation of B cells. This is particularly desirable for thetreatment of malignancies of B cell origin, such as leukemias andlymphomas. Such treatment also has the effect of sensitizing the cellsto ionizing radiation, so that the effect of ionizing radiation andphosphotyrosine phosphatase inhibitors are not additive but synergistic.

However, phosphotyrosine phosphatase inhibitors can also be used tocontrol proliferation of normal B cells, particularly in situations inwhich downregulation of the immune response is desired. Such situationsinclude induction of immunosuppression to prevent transplant rejection,as well as in the treatment of autoimmune diseases such as rheumatoidarthritis, Hashimoto's thyroiditis, and systemic lupus erythematosus, aswell as other autoimmune diseases.

Phosphotyrosine is also involved in proliferation of protozoans, such asamoebae and trypanosomes, a number of species of which are seriousparasites. Accordingly, such phosphotyrosine phosphatase inhibitors canalso be useful in treating protozoan-based diseases. It is predictedthat developmentally stage-specific tyrosine phosphorylation isdisrupted in these organisms. This disruption is predicted to lead todeath of the protozoa (M. Parsons et al., "Distinct Patterns of TyrosinePhosphorylation During the Life Cycle of Trypanosoma brucei," Molec.Biochem. Parasitol. 45:241-248 (1990).

The method of inhibiting B cell proliferation comprises the step ofcontacting proliferating B cells with a phosphotyrosine phosphataseinhibitor as described above. The compound is administered in a quantitysufficient to detectably inhibit proliferation of the cells as measuredby incorporation of nucleotides into DNA. The term "detectably inhibitproliferation," as used herein, refers to a detectable decrease ineither DNA synthesis or cell number, inasmuch as cell division followsDNA synthesis. Typically, the dose required is in the range of 1 μM to100 μM, more typically in the range of 5 μM to 25 μM. The exact doserequired can be readily determined from in vitro cultures of the cellsand exposure of the cells to varying doses of the phosphotyrosinephosphatase inhibitor. The effect of the phosphotyrosine phosphataseinhibitor can be judged by clonogenic assays, assays measuring theincorporation of radioactively labelled nucleotides into DNA, or otherassays measuring cell proliferation.

B. Treatment of Lymphoproliferative Disorders

Because of the effect of phosphotyrosine phosphatase inhibitors onproliferation of cells, particularly B cells, but also myeloid cells,such inhibitors can be used in a method of treating malignantproliferative disorders. The diseases that can be treated includeleukemias and lymphomas, and the proliferating cells can be either Bcells, or, alternatively, myeloid cells. The method comprises the stepof contacting the proliferating malignant cells with a phosphotyrosinephosphatase inhibitor as described above. The compound is administeredin a quantity sufficient to significantly inhibit proliferation of themalignantly proliferating cells, as that term is defined above. Thedosage range in general will be as described above. Further guidance forthe dosage is given in the Examples below.

The compositions can be administered using conventional modes ofadministration including, but not limited to, intravenous,intraperitoneal, oral, or intralymphatic. Oral or intraperitonealadministration is generally preferred.

The compositions can be in a variety of dosage forms which include, butare not limited to, liquid solutions or suspensions, tablets, pills,powders, suppositories, polymeric microcapsules or microvesicles,liposome, and injectable or infusible solutions. The preferred formdepends on the mode of administration and the quantity administered.

The compositions for administration including the phosphotyrosinephosphatase inhibitors preferably also include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchanges, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thephosphotyrosine phosphatase inhibitors as used in the methods of thepresent invention depend on the severity and course of the disease, thepatient's health, the response to treatment, the particular type ofmalignantly proliferating cells characteristic of the particularleukemia or lymphoma, pharmacokinetic considerations such as thecondition of the patient's liver and/or kidneys that can affect themetabolism and/or excretion of the administered phosphotyrosinephosphatase inhibitors, and the judgment of the treating physician.Accordingly, the dosages should be titrated to the individual patient.Nevertheless, an effective dose of the phosphotyrosine phosphataseinhibitors for use in the treatment methods of the present invention canbe in the range of from about 1 μM to about 100 μM in the blood and/ortissues.

Preferably, the dose of phosphotyrosine phosphatase inhibitor used issufficient to induce apoptosis in the malignantly proliferating cells.

The treatment method can further comprise a step of delivering ionizingradiation to the cells contacted with the phosphotyrosine phosphataseinhibitor. The ionizing radiation is delivered in a dose sufficient toinduce a substantial degree of cell killing among the malignantlyproliferating cells, as judged by assays measuring viable malignantcells. The degree of cell killing induced is substantially greater thanthat induced by either the phosphotyrosine phosphatase inhibitor aloneor the ionizing radiation alone. Typical forms of ionizing radiationinclude beta rays, gamma rays, alpha particles, and X-rays. These can bedelivered from an outside source, such as an X-ray machine or a gammacamera, or delivered to the malignant tissue from radionuclidesadministered to the patient. The use of radionuclides is well understoodin the art and need not be detailed further. A range of dosages that canbe used is between about 1 and 500 cGy (i.e., from about 1 to about 500rads).

C. Inhibition of Phosphotyrosine Phosphatases

Methods according to the present invention can also be used to inhibitphosphotyrosine phosphatases for purposes other than that of treatingmalignant disease. In particular, phosphotyrosine phosphatase inhibitorscan be used to suppress the immune system in order to prevent organ ortissue rejection during transplantation and also in the treatment ofautoimmune diseases such as rheumatoid arthritis, Hashimoto'sthyroiditis, systemic lupus erythematosus, Guillain-Barre Syndrome, andpossibly multiple sclerosis.

Methods according to the present invention can also be used in thetreatment of protozoan infections, such as amoebae and trypanosomes,whose proliferation also depends on phosphorylation.

Additionally, methods according to the present invention can be usefulin the study of B cells in culture, in order to determine theirsusceptibility to ionizing radiation and other reagents, such asalkylating agents and intercalating agents, that can interfere with DNAsynthesis. In particular, methods according to the present invention canbe used to block B cell development in vitro in order to determine theeffect of inhibition of proliferation of B cells on development ofimmune responses. For example, such methods could be used to augmentsignals and enhance calcium levels in cells (see Example 8). Suchmethods can also be used to block the effects of CD28. Accordingly, suchmethods can therefore be used to screen B cell cultures for abnormalUV-induced signals. Such screening could be clinically useful indetermining susceptibility to conditions related to ultravioletexposure, such as skin cancer.

D. Prevention of Class-Switching in Antibodies

Methods according to the present invention can further be used toprevent class-switching in antibodies from IgG or IgM to IgE. It isdesirable to block IgE production because this type of antibody mediatesmany allergic responses, particularly immediate-type hypersensitivityreactions such as anaphylaxis, atopy, and urticaria. The CD40 ligandgp39 and the cytokine IL-4 act on B cells to induce the switching of thetype of antibody produced from IgM to IgE. CD40 and IL-4 mechanisms ofaction are known to involve tyrosine phosphorylation. Phosphotyrosinephosphatase inhibitors such as BMLOV disrupt the normal pattern oftyrosine phosphorylation, disrupting the class-switching process. Theadministration of BMLOV, in particular, can markedly inhibit theproduction of IgE antibody while much less markedly inhibiting theproduction of IgG subclasses such as IgG1 and IgG4. This leads to theresult that the ratio of IgG to IgE increases (Example 14). This resultleads to the conclusion that phosphotyrosine phosphatase inhibitors suchas BMLOV have value in the treatment of allergy.

Accordingly, a method of preventing the class-switching ofantibody-producing cells comprises administering to antibody-producingcells a quantity of a phosphotyrosine phosphatase inhibitor sufficientto detectably reduce the production of IgE antibody by the cells.Typically, the cells also produce IgG antibody, and the quantity of thephosphotyrosine phosphatase inhibitor is such that the ratio of thequantity of IgG antibody produced by the cells to the quantity of IgEantibody produced by the cells increases.

Any of the phosphotyrosine phosphatase inhibitors disclosed above can beused to prevent class-switching, including metal-organic coordinatecovalent compounds, nonhydrolyzable phosphotyrosine analogs,dephostatin, and 4-(fluoromethyl)phenyl phosphate and its esterifiedderivatives. However, a preferred phosphotyrosine phosphatase inhibitorfor preventing class-switching is BMLOV.

The invention is illustrated by the following Examples. The Examples arefor illustrative purposes only and are not intended to limit theinvention.

EXAMPLES Example 1

Inhibition of Phosphotyrosine Phosphatases by Bis(Maltolato)Oxovanadium(IV) (BMLOV)

Bis(maltolato)oxovanadium (IV) (BMLOV) was synthesized as described inJ. H. McNeill et al., "Bis(maltolato)oxovanadium (IV) is a PotentInsulin Mimic," J. Med. Chem. 35:1489-1491 (1992). Briefly, the compoundwas synthesized by combining maltol (3-hydroxy-2-methyl-4-pyrone) andvanadyl sulfate in a 2:1 ratio in water. The pH of the solution wasraised to 8.5, and the solution was refluxed overnight. A deeppurple-green birefringent compound precipitated on cooling and wascollected. This compound is the BMLOV.

BMLOV was then assayed for its activity in inhibiting severalphosphatases, including phosphotyrosine phosphatases PTP1B and CD45, aswell as serine/threonine phosphatases PP1 and PP2A and calf intestinalalkaline phosphatase.

The tyrosine phosphatases PTP1B and CD45 were assayed withphosphorylated myelin basic protein as described in K. Guan et al.,Nature 350:359-362 (1991) except that 2-minute assays were performed.The PTP1B used was a GST fusion protein (Upstate Biotechnology, LakePlacid, N.Y.). The CD45 was immunoprecipitated from Jurkat T cells withthe monoclonal antibody 9.4 (ATCC No. HB 10270, deposited with theAmerican Type Culture Collection, Rockville, Md., on Oct. 20, 1989). Thephosphatases PP1 and PP2A (Upstate Biotechnology) and calf intestinalalkaline phosphatase (Sigma, St. Louis, Mo.) were assayed withp-nitrophenyl phosphate as a substrate.

The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Phosphatase Inhibition by BMLOV                                               Phosphatase   IC.sub.50, NM SEM    n                                          ______________________________________                                        PTPIB          26            7     3                                          CD45           25            1     3                                          PPI           6156          360    2                                          PP2A          3337          208    2                                          Alkaline Phosphatase                                                                        5 × 10.sup.5                                                                          --     2                                          ______________________________________                                    

BMLOV was found to be a potent inhibitor of PTP1B and CD45, both ofwhich are phosphotyrosine phosphatases. The drug showed substantialselectivity for the phosphotyrosine phosphatases relative to otherphosphatases. Much higher concentrations of the drug were required toinhibit the serine phosphatases PP1 and PP2A. Intestinal alkalinephosphatase was highly resistant to inhibition by BMLOV.

Example 2 Induction of Tyrosine Phosphorylation in TransformedLymphocytes

Ramos cells, a human B cell lymphoma cell line, were treated withvarying doses of BMLOV. The cells were lysed and lysates were subjectedto polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE). The resulting electropherograms were subjected toWestern blotting with anti-phosphotyrosine antibody to detect thepresence of phosphotyrosine in the cells.

The results are shown in FIGS. 1A-1C. FIG. 1A demonstrates that BMLOVinduces tyrosine phosphorylation in a dose-dependent manner when thecells were treated with varying doses for a period of 1 hour. FIG. 1Bshows the effects of treating cells with 100 μM BMLOV for varying times.The drug was found to begin to induce tyrosine phosphorylation in thecells by 8 minutes and the phosphorylation increased with furtherexposure.

FIG. 1C demonstrates that treatment of the cells for 16 hours with 50 μMBMLOV leads to high levels of tyrosine phosphorylation.

Example 3

Induction of Apoptosis by BMLOV

BMLOV was demonstrated to kill malignant leukemia and lymphoma cells byinducing apoptosis. Apoptosis is the process of programmed cell death.The hallmark of apoptosis is the fragmentation of DNA into fragmentswhose size distribution is observed as a ladder of bands on agarosegels. FIG. 2A demonstrates that BMLOV induced apoptosis in Ramos B celllymphoma cells by 48 hours at a dose of 10 μM, with apoptosis apparentat 24 hours with higher doses. Apoptosis was also observed to be inducedin the human acute promyelocytic leukemia cell line HL60 in a dosedependent manner (FIG. 2A). The induction of apoptosis was specific inthat apoptosis was not observed in the human T-cell leukemia cell linesCEM or Jurkat, nor was it observed in the human colon carcinoma cellline 3347 (FIG. 2B).

For these experiments, cell samples (2×10⁶ cells) were centrifuged at200×g, the media removed by aspiration and the cell pellets stored at-70° C. until processing. Cell pellets were resuspended in 300 μldigestion buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5%SDS, 0.1 mg/ml protease K) and incubated for 12 hours at 50° C.DNase-free ribonuclease (5 μg) in 200 μl TE buffer (10 mM Tris-HCl, pH7.5, 1 mM EDTA) was added to each sample followed by incubation for 2hours at 37° C. DNA was then extracted once with phenol:chloroform, oncewith chloroform and then precipitated with 0.5 volumes of ammoniumacetate and 2 volumes of ethanol at -70° C. for 12 hours. Theprecipitated DNA was resuspended in 200 μTE buffer and quantitated byabsorbance at 260 nanometers. The DNA (15 μg) was applied to a 1.3%agarose gel in TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA)resolved at 20 mA constant current and the DNA was visualized bystaining with ethidium bromide. Fragmented DNA due to apoptosis appearedas a ladder of bands on the gel.

Example 4 Selective Cytotoxicity of BMLOV as Examined in ClonogenicAssays

The selective cytotoxicity of BMLOV was examined in clonogenic assays.Cells were grown in methylcellulose media and the number of coloniesformed after seven days of treatment with various doses of the drug weredetermined. The clonogenic assays were performed with six replicates foreach treatment as described F. M. Uckun et al., J. Exp. Med. 163:347-368(1986); F. M. Uckun & J. A. Ledbetter, Proc. Natl. Acad. Sci. USA85:8603-8607 (1988)). The data is given plus/minus the standard error ofthe mean.

The data is shown in Table 2. The three B-cell lines, Ramos, Raji, andREH, were all highly sensitive to doses of 5 to 10 μM BMLOV. The myeloidcell line THP-1 and the promyelocytic cell line HL-60 also were highlysensitive to BMLOV at a dose of only 1 μM. BMLOV gave 99.8% clonogeniccell death for the Raji transformed B cell line at a dose of 10 μM, and99.4% clonogenic cell death for HL-60 promyelocytic leukemia cells at 1μM.

                                      TABLE 2                                     __________________________________________________________________________    Inhibition of Leukemia and Lymphoma Cell Line Growth                          by BMLOV as Measured in Clonogenic Assays                                     Cell Line                                                                     Dose, μM                                                                        Ramos Raji  REH    THP-1 HL-60                                           __________________________________________________________________________    0    4065 ± 840                                                                       3252 ± 672                                                                       5122 ± 1296                                                                       6453 ± 1563                                                                      5122 ± 1241                                  1.0  179 ± 33                                                                         1291 ± 283                                                                       1810 ± 385                                                                        1613 ± 235                                                                       31 ± 8                                            (95.6%)                                                                             (60.3%)                                                                             (64.7%)                                                                              (75.0%)                                                                             (99.4%)                                         5.0   7 ± 2                                                                            4 ± 0.6                                                                         71 ± 8                                                                            127 ± 35                                                                         31 ± 8                                            (99.8%)                                                                             (99.9%)                                                                             (98.6%)                                                                              (98.0%)                                                                             (99.4%)                                         10.0 0      3 ± 0.8                                                                         8 ± 1                                                                             127 ± 35                                                                         15 ± 4                                                  (99.9%)                                                                             (99.8%)                                                                              (98.0%)                                                                             (99.7%)                                         __________________________________________________________________________

Data is given in colonies per 10⁴ cells; the percentage of inhibition isshown in parentheses.

By contrast, the T cell line CEM or MDAMB-453 breast carcinoma cellswere inhibited only moderately by a dose as high as 100 μM of BMLOV.

Example 5 The Effect of BMLOV on Incorporation of Nucleotides into DNA

In order to determine the effects of BMLOV on various cell types,including a number of cell types that did not grow well in themethylcellulose clonogenic assay, the cells were grown for seven days inthe presence of BMLOV. For each cell type, the cells were passageduniformly for all drug doses given during the seven-day period. Thecells were then pulsed for six hours with ³ H!thymidine. The extent ofDNA synthesis was then determined by counting the radioactivityincorporated into the cells. The data is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Inhibition of Leukemia and Lymphoma Cell Line Growth                          by BMLOV as Measured in Thymidine Incorporation Assays                        Dose,                                                                               .sup.3 H! cpm Incorporation for Cell Line:                              μM                                                                              Ramos   BCL.sub.1                                                                             A20   THP-1 HL-60 Jurkat                                                                              CEM                              ______________________________________                                         0   292342  271144  389480                                                                              202301                                                                              249183                                                                              143917                                                                              395157                            1   289936  265036  419947                                                                              201670                                                                              191637                                                                              149950                                                                              368721                            5   65545   226714  289932                                                                              178290                                                                              276505                                                                              118720                                                                              403576                           10   167     55396   66157 199993                                                                              222178                                                                              98057 371256                           25   149     195     281   112209                                                                              11398 26511 313714                           ______________________________________                                    

Ramos cells were highly sensitive to doses of 10 and 25 μm BMLOV. Themurine cell line BCL₁ is a highly tumorigenic and lethal leukemiaconsidered to be a model of human chronic lymphocytic leukemia (CLL).These cells were highly sensitive to the drug at a dose of 25 μM. THP-1cells showed only partial sensitivity to the drug at a dose of 25 μM. Ingeneral, cells were more sensitive to the drug in the clonogenic assaysthan in the thymidine incorporation assay. These results suggest agreater requirement for phosphatase activity for cells to grow ascolonies in methylcellulose relative to growth in free suspension inliquid media. The murine B cell lymphoma line A20, which forms highlyaggressive tumors in mice, was very sensitive to a dose of 25 μM of thedrug. The human promyelocytic leukemia cell line HL60 and the humanacute T cell leukemia cell line Jurkat were moderately sensitive toBMLOV at a dose of 25 μM, whereas the human T cell acute lymphocyticleukemia CEM was resistant. These results indicate that malignant cellsof B cell origin, including lymphoma, acute lymphocytic leukemia, andchronic lymphocytic leukemia are sensitive to BMLOV. Some leukemias ofmyeloid and T cell origin also show sensitivity to BMLOV whereas othersare resistant.

Example Effect of BMLOV on Normal B Cell Proliferation

BMLOV can inhibit normal tonsillar B cell proliferation driven bystimulation of CD40 via either anti-CD40 antibody or gp39 ligand pluseither anti-CD20 antibody or phorbol 12-myristate 13-acetate (PMA). Inone experiment, doses of 0.1 to 10 μM had little effect on proliferation(FIG. 3). In a second experiment, a dose of 5 μM gave substantialinhibition of proliferation and a dose of 50 μM completely blockedproliferation (FIG. 4). Variations between the individuals from whichthe tonsils were derived could account for the differences between theseexperiments.

Similarly, BMLOV can inhibit the proliferation of normal peripheral Bcells. Normal B cell proliferation is mediated in part by the B cellsurface molecule CD20, the B cell surface molecule CD40 in conjunctionwith its ligand gp39, and by the cytokine IL-4. The pharmacologic agentphorbol 12-myristate 13-acetate (PMA) can also be used in combinationwith these biological stimulation agents to further increaseproliferation.

In the experiment reported in Table 4, monoclonal antibodies to CD20 andCD40 were used to stimulate proliferation. Peripheral B cells wereisolated from two healthy human volunteers. The cells were stimulated aslisted in Table 4 and the effects of various doses of BMLOV onproliferation, as measured by ³ H!thymidine incorporation, weredetermined. BMLOV was able to inhibit proliferation induced by CD20,CD40, IL-4 and PMA in the various combinations tested. The cells fromdonor 2 were more sensitive, indicating some variation among individualsin their sensitivity to the drug.

It is important to note that the extent to which the proliferation ofnormal B cells was inhibited in this example (Table 4) was less than forthe B cell leukemia and lymphoma cells examined in Examples 4 and 5.Phosphotyrosine phosphatase inhibitors such as BMLOV thus have thepotential to act more selectively on malignant B cells than on normal Bcells, offering an important advantage in the treatment of leukemia andlymphoma.

                  TABLE 4                                                         ______________________________________                                        INHIBITION OF NORMAL PERIPHERAL B CELL GROWTH BY                              BMLOV AS MEASURED IN  .sup.3 H!-THYMIDINE INCORPORATION                       ASSAYS                                                                         .sup.3 H! cpm Incorporation For Stimuli:                                     Dose, μm                                                                          PMA + CD40 CD20 + CD40                                                                              PMA + IL4                                                                             CD40 + IL4                               ______________________________________                                        Donor #1:                                                                      0     65587      3574       61941   32499                                     1     62643      3619       61415   31966                                     5     58418      3644       48724   33845                                    10     45330      3422       45278   31637                                    25     25536      2432       14277   24933                                    Donor #2:                                                                      0     52672      5602       62228   19095                                     1     49102      4593       53642   16597                                     5     47624      5681       41892   18824                                    10     29024      5269       20313   12662                                    25     15671       856        2730    6013                                    ______________________________________                                    

Standard error did not exceed 8% for donor 1 and 15% for donor 2.Stimulation of CD20 was via monoclonal antibody 1F5 and stimulation ofCD40 was via monoclonal antibody G28-5.

Example 7 Sensitization of Ramos B Cells to Ionizing Radiation by BMLOV

Ionizing radiation in conjunction with bone marrow transplantation is amajor therapy for leukemia. However, many leukemias are resistant toradiation, limiting the efficacy of this therapy.

It was previously demonstrated that ionizing radiation stimulatestyrosine kinases in human B lymphocyte precursors, triggering apoptosisand clonogenic cell death, an effect that was markedly enhanced by thephosphatase inhibitor orthovanadate.

In clonogenic assays, as shown in FIG. 5, the combination of 1 μM BMLOVwith 265 cGy radiation gave a 10-fold increase in cell death relative toradiation alone. The combination of 2μM BMLOV with 265 cGy radiationgave a 50-fold increase in clonogenic cell death relative to radiationalone. These effects were synergistic rather than additive in that anapproximately 5-fold enhancement in sensitization for the combinationtreatment was observed relative to expected additive effects based onthe use of either treatment alone. These results suggest thatphosphatase inhibitors such as BMLOV can be of value for use incombination with radiation therapy for the treatment of B cellmalignancies.

Example 8 Inhibition by BMLOV of CD28-Mediated Ca²⁺ Signals and CellProliferation

BMLOV reproducibly caused marked inhibition of CD28 Ca²⁺ signals in PHAT cell blasts. The inhibition was specific in that such inhibition wasnot observed for signals generated by CD3 alone or in combination withCD2 or CD4.

PBL growth driven by anti-CD28 antibody plus interleukin-2 (IL-2) wasmarkedly inhibited by 1 μM BMLOV (FIG. 6) and growth driven by anti-CD3antibody plus anti-CD28 antibody was strongly inhibited by 10 μM BMLOV(FIG. 7).

In contrast, substantially less inhibition was observed for IL-2 alone,PMA in combination with anti-CD28, or the factor-independent growth ofthe T cell line CEM, indicating the specificity of these effects.

Mixed lymphocyte response cultures that are dependent on CD28costimulation were inhibited by over 50% by 0.5 to 5 μM BMLOV (FIG. 8).These results suggest the phosphatase inhibitor may selectively blockCD28 effects and therefore can be used to block the generation ofantibodies to antigens in animals.

An important question has been how does the costimulatory CD28 signaldiffer from the primary CD3 dependent signal in T cells, since bothsignals induce tyrosine phosphorylation and Ca²⁺ flux. These resultsraise the possibility that the CD28 costimulatory or second signalrequires a BMLOV-sensitive phosphatase activity that the primary signalsdo not.

Example 9 Augmentation of Signals in T and B Cells by BMLOV

BMLOV was found to markedly enhance basal calcium levels in PHA blastswhen the cells were treated with doses of 50 to 100 μM for fifteenhours. These results indicate that phosphatase activity is required tomaintain normal calcium levels in the cell. Treatment of the cells with25-50 μM BMLOV was found to prolong Ca²⁺ signals generated bycrosslinking CD3 alone or in combination with CD2 and CD4. BMLOV greatlyincreased and prolonged UV-induced Ca²⁺ signals in Ramos B cells,indicating that phosphatase activity may limit UV-induced signals. Incontrast, BMLOV did not alter Ca²⁺ signals induced in Ramos B cells bycrosslinking sIg. However, BMLOV enhanced the tyrosine phosphorylationresponse of Ramos B cells to sIg crosslinking, particularly at earlytime points. These results suggest a potential for BMLOV to augment somecell responses.

Example 10 BMLOV Inhibits Src-Family Kinase Activity in Lymphocytes andColon Carcinoma Cells

The Src-family kinases are known to require cellular phosphotyrosinephosphatase activity in order for them to respond in biologicallystimulated cells. This is because Src-family kinases require aC-terminal tyrosine phosphorylation site to be dephosphorylated foractivation to occur. Phosphotyrosine phosphatase inhibitors maytherefore be expected to inhibit Src kinases indirectly by preventingtheir activation.

Treatment of Ramos cells with BMLOV for 15 hours inhibited the activityof the Src kinases Lyn and Fyn from the cells by approximately 50%.

Many colon carcinomas are known to express the Src oncogene product athigh levels. In two colon carcinoma cell lines expressing Src,pp60^(c-src) activity was strongly inhibited by BMLOV in adose-dependent fashion (FIG. 9), while pp60^(c-src) protein levelremained constant. The treated cells showed morphological changes,including decreased adherence.

Example 11 BMLOV Alters the Cell Cycle Progression of Treated Cells

A variety of anti-cancer therapies are known to alter the cell cycleprogression of tumor cells. For example, both cisplatin and radiationtherapy result in accumulation of cells in G2/M phase.

Ramos B cells treated with various concentrations of BMLOV for 16 hourswere examined for their DNA content by propidium diiodide stainingfollowed by FACScan flow cytometric analysis using the SFIT program.

The results are shown in FIG. 10. The percentages of cells in G1, S, andG2/M are listed for doses of 0 to 5 μM of BMLOV. Percentages could notbe calculated for higher doses due to the strong effects of the drug incausing apoptosis. Apoptotic cells lacking normal amounts of DNA aremarked "A" in the 5 μM dose panel. BMLOV was found to preferentiallydeplete the proportion of cells in G1 while increasing the proportion inS phase. Loss of DNA due to apoptotic cell death was readily apparent ina dose of 5 μM and was greatly increased at high doses.

Example 12 Effects of Analogs of BMLOV

Two analogs of BMLOV were prepared, vanadyl 1-benzoyl acetonate andvanadyl 2-acetyl 1-tetralonate.

The synthesis of vanadyl 2-acetyl 1-tetralonate was as follows. Aquantity of 2-acetyl 1-tetralone (1.13 g; 0.006 moles) was dissolved in74 ml of water with the pH adjusted to 13 with NaOH. A small amount ofinsoluble material has removed. Vanadyl sulfate (0.003 moles) was thenadded. The solution was then heated and allowed to reflux for 1 hour.The solution was then cooled on ice and 0.884 g product (a green solid)was collected and dried over P₂ O₅. The yield was 33%. A similarprocedure was used to synthesize vanadyl 1-benzoyl acetonate, startingwith 1-benzoyl acetone. Vanadyl acetylacetonate, available commercially,was also studied.

Of these analogs, only vanadyl acetylacetonate and vanadyl 1-benzoylacetonate strongly induced cellular tyrosine phosphorylation. As shownin FIG. 11, anti-phosphotyrosine western blot analysis of whole celllysates following SDS-PAGE revealed that vanadyl acetylacetonatestrongly induced tyrosine phosphorylation in a pattern very similar tothat of BMLOV.

Analogs of vanadyl acetylacetonate were prepared in which the vanadiumwas replaced with other metals, namely molybdenum, chromium, iron,manganese, or copper. Of these analogs with differing metal ions, onlycupric acetylacetonate induced tyrosine phosphorylation, but in adifferent pattern than for the vanadyl compounds (FIG. 11). Thus, onlycertain metals are active in this type of compound.

Clonogenic assays of Ramos Burkitt lymphoma cells treated with cupricacetylacetonate showed that the cells were sensitive to the compound atdoses of 10 and 25 μM (Table 5).

                  TABLE 5                                                         ______________________________________                                        Effect of Cupric Acetylacetonate on Ramos B Cells as                          Measured in Clonogenic Assays                                                 Dose, μM     Colonies SE                                                   ______________________________________                                        0               9126     1944                                                 1               5749     1070                                                 5                226      26                                                  10               127      35                                                  25               31        8                                                  ______________________________________                                    

Example 13 BMLOV Was Tolerated by Animals

BMLOV is tolerated by animals when it was administered by oral orintraperitoneal routes. Mice were treated with 1.6 mM of BMLOV in theirdrinking water continuously for 10 days, and the level of the drug intheir blood serum was determined by atomic absorption spectroscopy after1 and 10 days of treatment, as shown in Table 6. The mice displayed noovert ill effects from the drug treatment. Subsequently, mice havereceived intraperitoneal doses of BMLOV of up to 1 mg and oral doses ofup to 1.6 mg without ill effects except for some temporary lethargy.

                  TABLE 6                                                         ______________________________________                                        BMLOV Levels in Mouse Sera                                                    Mouse       Day    BMLOV in Serum, μM                                      ______________________________________                                        647          1     9.5                                                        648          1     5.3                                                        649          1     7.5                                                        650          1     8.7                                                        647         10     11.4                                                       648         10     11.0                                                       649         10     7.5                                                        650         10     7.7                                                        ______________________________________                                    

Example 14 Prevention of Class-Switching in Antibody-Producing B Cellsby BMLOV

The effects of BMLOV were assayed on class-switching inantibody-producing B cells. Human B cells producing antibody weretreated with anti-CD40 antibody plus IL-4, which increased production ofIgE over 10-fold (Table 7). However, in the presence of 5.6 or 17 μMBMLOV, the increased production of IgE was markedly inhibited. Incontrast, the production of IgG1 and IgG4 was much less affected,particularly at a dose of 5.6 μM BMLOV. This selective effect isimportant because the IgG antibody production is an important responseto infectious disease. It would be of value to suppress IgE productionfor the treatment of allergies while maintaining IgG production,particularly in conditions in which an allergy coexists with aninfectious disease. A common example is the exacerbation of allergicrhinitis (hay fever) as the result of a respiratory infection.

                  TABLE 7                                                         ______________________________________                                        EFFECT OF BMLOV ON CLASS-SWITCHING IN                                         ANTIBODY-PRODUCING B CELLS                                                    Treatment of      Ig!, ng/ml:                                                 Cells            IgGl   Ig64     IgM  IgE                                     ______________________________________                                        Untreated        225    0.7      6.6  0.5                                     Anti-CD40 + IL-4 510    1.7      16.8 5.7                                     Anti-CD40 +      450    1.7      20.4 3.9                                     IL-4 + 0.002 μM BMLOV                                                      Anti-CD40 +      450    1.1      20.4 2.6                                     IL-4 + 0.008 μM BMLOV                                                      Anti-CD40 +      390    1.3      19.2 4.5                                     IL-4 + 0.02 μM BMLOV                                                       Anti-CD40 +      450    2.7      12.0 5.1                                     IL-4 + 0.07 μM BMLOV                                                       Anti-CD40 +      495    1.2      13.8 7.5                                     IL-4 + 0.2 μM BMLOV                                                        Anti-CD40 +      480    1.3      17.4 5.7                                     IL-4 + 0.6 μM BMLOV                                                        Anti-CD40 +      450    2.7      15.6 3.0                                     IL-4 + 1.9 μM BMLOV                                                        Anti-CD40 +      480    1.8      18.6 1.2                                     IL-4 + 5.6 μM BMLOV                                                        Anti-CD40 +      450    0.8      18.0 0.8                                     IL-4 + 17 μM BMLOV                                                         Anti-CD40 +       60    0.5      5.1  0.2                                     IL-4 + 50 μM BMLOV                                                         ______________________________________                                    

ADVANTAGES OF THE INVENTION

The present invention provides methods for inhibiting phosphotyrosinephosphatase, particularly in B cells. This yields improved methods ofinhibiting the proliferation of these cells by exploiting the occurrenceof apoptosis (programmed cell death). These methods can be exploited fortreatment of disorders marked by malignant proliferation of B cells,such as leukemias and lymphomas, and can be combined with other methodsof treatment, including radiation. Such a combination yields synergisticeffects over either radiation alone or the use of phosphotyrosinephosphatase inhibitors alone.

Methods according to the present invention can also be used forcontrolling proliferation of non-malignant B cells for regulation of theimmune response. This is desirable for the treatment of autoimmunedisease and for controlling transplant rejection, as well as forcontrolling class-switching in antibody-producing cells.

The methods of the present invention are further useful for studyingsignaling in B cells and for screening for abnormalities of B cellsignaling.

Although the present invention has been described with considerabledetail, with reference to certain preferred versions thereof, otherversions are possible. Therefore, the spirit and scope of the appendedclaims should not be limited to the description of the preferredversions contained herein.

I claim:
 1. A method of inhibiting B cell proliferation comprising thestep of contacting proliferating B cells with a nonhydrolyzablephosphotyrosine analog selected from the group consisting of N-arylphosphoramidates, N-aryl phosphorothioates, and N-aryl phosphonates, inwhich the aryl moiety is optionally substituted at any of the ortho,meta, and para positions, and one or two of the oxygen atoms bound tothe phosphorus are optionally esterified, the nonhydrolyzablephosphotyrosine analog being administered in a quantity sufficient todetectably inhibit proliferation as measured by incorporation ofnucleotides in DNA.
 2. The method of claim 1 wherein the compoundinduces apoptosis in the proliferating B cells.
 3. The method of claim 1wherein the nonhydrolyzable phosphotyrosine analog is a N-arylphosphoramidate of formula XVI, in which each of R₁ through R₇ isselected from the group consisting of hydrogen and C₁ -C₅ alkyl##STR29##
 4. The method of claim 3 wherein each of R₁ through R₅ ishydrogen.
 5. The method of claim 3 wherein at least one of R₆ and R₇ isother than hydrogen.
 6. The method of claim 1 wherein thenonhydrolyzable phosphotyrosine analog is a phosphorothioate of formulaXVII in which each of R₁ through R₇ is selected from the groupconsisting of hydrogen and C₁ -C₅ alkyl ##STR30##
 7. The method of claim6 wherein each of R₁ through R₅ is hydrogen.
 8. The method of claim 7wherein at least one of R₆ and R₇ is hydrogen.
 9. The method of claim 1wherein the nonhydrolyzable phosphotyrosine analog is a phosphonate offormula XVIII in which each of R₁ through R₇ is selected from the groupconsisting of hydrogen and C₁ -C₅ alkyl ##STR31##
 10. The method ofclaim 9 wherein each of R₁ through R₅ is hydrogen.
 11. The method ofclaim 10 wherein at least one of R₆ and R₇ is other than hydrogen.
 12. Amethod of inhibiting phosphotyrosine phosphatase in proliferating Bcells comprising the step of contacting proliferating B cells with anonhydrolyzable phosphotyrosine analog selected from the groupconsisting of N-aryl phosphoramidates, N-aryl phosphorothioates, andN-aryl phosphonates, in which the aryl moiety is optionally substitutedat any of the ortho, meta, and para positions, and one or two of theoxygen atoms bound to the phosphorus are optionally esterified, thenonhydrolyzable phosphotyrosine analog being administered to the B cellsin a quantity sufficient to inhibit the activity of phosphotyrosinephosphatase in the cells.
 13. The method of claim 12 wherein thenonhydrolyzable phosphotyrosine analog is a N-aryl phosphoramidate offormula XVI, in which each of R₁ through R₇ is selected from the groupconsisting of hydrogen and C₁ -C₅ alkyl ##STR32##
 14. The method ofclaim 13 wherein each of R₁ through R₅ is hydrogen.
 15. The method ofclaim 14 wherein at least one of R₆ and R₇ is other than hydrogen. 16.The method of claim 12 wherein the nonhydrolyzable phosphotyrosineanalog is a phosphorothioate of formula XVII in which each of R₁ throughR₇ is selected from the group consisting of hydrogen and C₁ -C₅ alkyl##STR33##
 17. The method of claim 16 wherein each of R₁ through R₅ ishydrogen.
 18. The method of claim 17 wherein at least one of R₆ and R₇is other than hydrogen.
 19. The method of claim 12 wherein thenonhydrolyzable phosphotyrosine analog is a phosphonate of formula XVIIIin which each of R₁ through R₇ is selected from the group consisting ofhydrogen and C₁ -C₅ alkyl ##STR34##
 20. The method of claim 19 whereineach of R₁ through R₅ is hydrogen.
 21. The method of claim 20 wherein atleast one of R₆ and R₇ is other than hydrogen.
 22. A method of treatinga subject suffering from a malignant proliferative disorder selectedfrom the group consisting of leukemias and lymphomas wherein theproliferating cells are selected from the group consisting of B cellsand myeloid cells, the method comprising the step of contacting theproliferating malignant cells with a nonhydrolyzable phosphotyrosineanalog selected from the group consisting of N-aryl phosphoramidates,N-aryl phosphorothioates, and N-aryl phosphonates, in which the arylmoiety is optionally substituted at any of the ortho, meta, and parapositions, and one or two of the oxygen atoms bound to the phosphorusare optionally esterified, the nonhydrolyzable phosphotyrosine analogbeing administered in a quantify sufficient to significantly inhibitproliferation of the malignantly proliferating cells.
 23. The method ofclaim 22 wherein the nonhydrolyzable phosphotyrosine analog is a N-arylphosphoramidate of formula XVI, in which each of R₁ through R₇ isselected from the group consisting of hydrogen and C₁ -C₅ alkyl##STR35##
 24. The method of claim 23 wherein each of R₁ through R₅ ishydrogen.
 25. The method of claim 24 wherein at least one of R₆ and R₇is other than hydrogen.
 26. The method of claim 22 wherein thenonhydrolyzable phosphotyrosine analog is a phosphorothioate of formulaXVII in which each of R₁ through R₇ is selected from the groupconsisting of hydrogen and C₁ -C₅ alkyl ##STR36##
 27. The method ofclaim 26 wherein each of R₁ through R₅ is hydrogen.
 28. The method ofclaim 27 wherein at least one of R₆ and R₇ is other than hydrogen. 29.The method of claim 22 wherein the nonhydrolyzable phosphotyrosineanalog is a phosphonate of formula XVIII in which each of R₁ through R₇is selected from the group consisting of hydrogen and C₁ -C₅ alkyl##STR37##
 30. The method of claim 29 wherein each of R₁ through R₅ ishydrogen.
 31. The method of claim 30 wherein at least one of R₆ and R₇is other than hydrogen.
 32. A method of treating a subject sufferingfrom a malignant proliferative disorder selected from the groupconsisting of leukemias and lymphomas wherein the proliferating cellsare selected from the group consisting of B cells and myeloid cells, themethod comprising the steps of:(a) administering a nonhydrolyzablephosphotyrosine analog selected from the group consisting of N-arylphosphoramidates, N-aryl phosphorothioates, and N-aryl phosphonates, inwhich the aryl moiety is optionally substituted at any of the ortho,meta, and para positions, and one or two of the oxygen atoms bound tothe phosphorus are optionally esterified, the nonhydrolyzablephosphotyrosine analog being administered in a quantity sufficient todetectably inhibit proliferation of the malignantly proliferating cells;and (b) delivering ionizing radiation to the cells contacted with thenonhydrolyzable phosphotyrosine analog, the ionizing radiation beingdelivered in a dose sufficient to induce a substantial degree of cellkilling among the malignantly proliferating cells, the degree of cellkilling induced being substantially greater than that induced by eitherthe nonhydrolyzable phosphotyrosine analog or the ionizing radiationalone.
 33. The method of claim 32 wherein the nonhydrolyzablephosphotyrosine analog is a N-aryl phosphoramidate of formula XVI, inwhich each of R₁ through R₇ is selected from the group consisting ofhydrogen and C₁ -C₅ alkyl ##STR38##
 34. The method of claim 33 whereineach of R₁ through R₅ is hydrogen.
 35. The method of claim 34 wherein atleast one of R₆ and R₇ is other than hydrogen.
 36. The method of claim32 wherein the nonhydrolyzable phosphotyrosine analog is aphosphorothioate of formula XVII in which each of R₁ through R₇ isselected from the group consisting of hydrogen and C₁ -C₅ alkyl##STR39##
 37. The method of claim 36 wherein each of R₁ through R₅ ishydrogen.
 38. The method of claim 37 wherein at least one of R₆ and R₇is other than hydrogen.
 39. The method of claim 32 wherein thenonhydrolyzable phosphotyrosine analog is a phosphonate of formula XVIIIin which each of R₁ through R₇ is selected from the group consisting ofhydrogen and C₁ -C₅ alkyl ##STR40##
 40. The method of claim 39 whereineach of R₁ through R₅ is hydrogen.
 41. The method of claim 40 wherein atleast one of R₆ and R₇ is other than hydrogen.
 42. A method ofinhibiting B cell proliferation comprising the step of contactingproliferating B cells with a phosphotyrosine phosphatase inhibitorselected from the group consisting of:(a) dephostatin; and (b) anoptionally esterified 4-(fluoromethyl)phenyl phosphate of formula XX, inwhich R₁ and R₂ are independently selected from the group consisting ofhydrogen and C₁ -C₅ alkyl, the phosphotyrosine phosphatase inhibitorbeing administered in a quantity sufficient to detectably inhibitproliferation as measured by incorporation of nucleotides into DNA##STR41##
 43. The method of claim 42 wherein the phosphotyrosinephosphatase inhibitor is an esterified 4-(fluoromethyl)phenyl phosphateof formula XX in which at least one of R₁ and R₂ is C₁ -C₅ alkyl.
 44. Amethod of inhibiting phosphotyrosine phosphatase in proliferating Bcells comprising the step of contacting proliferating B cells with aphosphotyrosine phosphatase inhibitor selected from the group consistingof:(a) dephostatin; and (b) an optionally esterified4-(fluoromethyl)phenyl phosphate of formula XX, in which R₁ and R₂ areindependently selected from the group consisting of hydrogen and C₁ -C₅alkyl, the phosphotyrosine phosphatase inhibitor being administered tothe B cells in a quantity sufficient to inhibit the activity ofphosphotyrosine phosphatase in the cells ##STR42##
 45. The method ofclaim 44 wherein the phosphotyrosine phosphatase inhibitor is anesterified 4-(fluoromethyl)phenyl phosphate of formula XX in which atleast one of R₁ and R₂ is C₁ -C₅ alkyl.
 46. A method of treating asubject suffering from a malignant proliferative disorder selected fromthe group consisting of leukemias and lymphomas wherein theproliferating cells are selected from the group consisting of B cellsand myeloid cells, the method comprising the step of contacting theproliferating malignant cells with a phosphotyrosine phosphataseinhibitor selected from the group consisting of:(a) dephostatin; and (b)an optionally esterified 4-(fluoromethyl)phenyl phosphate of formula XX,in which R₁ and R₂ are independently selected from the group consistingof hydrogen and C₁ -C₅ alkyl, the phosphotyrosine phosphatase inhibitorbeing administered in a quantity sufficient to significantly inhibitproliferation of the malignantly proliferating cells ##STR43##
 47. Themethod of claim 46 wherein the phosphotyrosine phosphatase inhibitor isan esterified 4-(fluoromethyl)phenyl phosphate of formula XX in which atleast one of R₁ and R₂ is C₁ -C₅ alkyl.
 48. A method of treating asubject suffering from a malignant proliferative disorder selected fromthe group consisting of leukemias and lymphomas wherein theproliferating cells are selected from the group consisting of B cellsand myeloid cells, the method comprising the steps of:(a) administeringa phosphotyrosine phosphatase inhibitor selected from the groupconsisting of:(i) dephostatin; and (ii) an optionally esterified4-(fluoromethyl)phenyl phosphate of formula XX, in which R₁ and R₂ areindependently selected from the group consisting of hydrogen and C₁ -C₅alkyl, the phosphotyrosine phosphatase inhibitor being administered in aquantity sufficient to detectably inhibit proliferation of themalignantly proliferating cells; and (b) delivering ionizing radiationto the cells contacted with the phosphotyrosine phosphatase inhibitor,the ionizing radiation being delivered in a dose sufficient to induce asubstantial degree of cell killing among the malignantly proliferatingcells, the degree of cell killing induced being substantially greaterthan that induced by either the phosphotyrosine phosphatase inhibitor orthe ionizing radiation alone ##STR44##
 49. The method of claim 48wherein the phosphotyrosine phosphatase inhibitor is an esterified4-(fluoromethyl)phenyl phosphate of formula XX in which at least one ofR₁ and R₂ is C₁ -C₅ alkyl.
 50. A method of preventing theclass-switching of antibody-producing cells comprising administering toantibody-producing cells a quantity of a phosphotyrosine phosphataseinhibitor sufficient to detectably reduce the production of IgE antibodyby the cells.
 51. The method of claim 50 wherein the antibody-producingcells also produce IgG antibody, and the quantity of the phosphotyrosinephosphatase inhibitor is such that the ratio of the quantity of IgGantibody produced by the cells to the quantity of IgE antibody producedby the cells increases.
 52. The method of claim 50 wherein thephosphotyrosine phosphatase inhibitor is bis(maltolato)oxovanadium (IV).